The effect of ultrasonic pre-treatment on the catalytic activity of lipases in aqueous and non-aqueous media
© Shah et al; This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2008
Received: 27 October 2007
Accepted: 30 January 2008
Published: 30 January 2008
Ultrasound has been used to accelerate the rates of numerous chemical reactions, however its effects on enzymatic reactions have been less extensively studied. While known to result in the acceleration of enzyme-catalysed reactions, ultrasonication has also been shown to induce enzyme inactivation. In this study we investigated the effects of ultrasonic pretreatment on lipases in both aqueous and non-aqueous media.
Our results show that the ultrasonic pre-irradiation of lipases (from Burkholderia cepacia and Pseudomonas fluorescens) in aqueous buffer and organic solvents enhanced enzymic activities. In addition, we report the enhancement of hydrolytic (esterase) and transesterification activities.
On using pre-irradiated enzyme, we found that the conversion rate for the transesterification of ethyl butyrate to butyl butyrate, increased from 66% to 82%. Similarly, a 79% conversion of Jatropha oil to biodiesel was observed upon employing pre-irradiated enzyme, in contrast to a 34% conversion with untreated enzyme.
CD spectra showed that while the enzyme's secondary structure remained largely unaffected, the microenvironments of aromatic amino acids were altered, with perturbation of the tertiary structure having also occurred. SEM analysis demonstrated significant morphological changes in the enzyme preparation as a result of ultrasonication.
In contrast to the effects of ultrasonic irradiation on other enzymes, for the lipases focused upon in this study, we report an enhancement of biocatalytic activity, which is thought to originate from morphological changes on the macro and molecular levels.
Ultrasound has been used to accelerate the rates of numerous of chemical reactions [1–3]. These rate enhancements, mediated by cavitation, are believed to originate from the build up of high local pressures (up to 1000 atm) and temperatures (up to 5000 K), as well as increased catalytic surface areas.
The effects of ultrasound on enzymatic reactions, however, have been less extensively studied [4–9]. The few studies that have been carried out can be categorised into two main groups. The first approach involves using ultrasound as an enzymic pretreatment to reduce particle size. This is especially relevant when using enzyme powders to catalyse reactions in organic media [10–12]. In such cases, the reduction in particle size and consequent increase in the catalytic surface area are thought to reduce mass transfer limitations. The second approach involves the use of ultrasound throughout the reaction. Here the cavitation energy is thought to accelerate the reaction rate, yet the mechanism by which this occurs is unclear. It may be that by increasing the movement of liquid molecules, the substrate's access to the active site is increased. Other mechanisms have also been suggested .
While it has been shown that the second of these approaches can accelerate enzymatic reactions [4, 6], other reports have demonstrated enzyme inactivation [7–9]. In general, enzymes are known to be more stable in nearly-anhydrous organic solvents [10–12], therefore it is not surprising that all the reported cases of rate enhancements resulting from ultrasonic treatment are those involving enzymic catalysis in organic media [4, 6].
We placed lipases in both aqueous as well as organic media during ultrasonic irradiation. The pretreated lipases were evaluated for both esterase (in water) and transesterification (in organic solvent) activities. Lastly, ultrasonically pre-irradiated lipase was also used in the transesterification of Jatropha oil to biodiesel. Biodiesel production is an interesting application of lipase-catalysed transesterification [13, 14], for which Jatropha oil can be considered an economically sound choice as a starting material [14, 15].
Results and Discussion
The effect of ultrasonic pre-irradiation on the esterase activity of lipases
For enzymes suspended in organic solvents, the latter was removed by centrifugation. The centrifuged enzyme was then transferred to the aqueous assay (hydrolysis of p-nitrophenyl palmitate) system in which it dissolved as expected (note: there was no organic solvent present in the aqueous assay system). In all cases, enzymic activity was seen initially to increase before then decreasing as a result of ultrasonic pre-irradiation (Figure 1). The effects were marginal for enzyme suspensions in organic solvents, but were quite significant for those in aqueous solution. The likeliest explanation for the trend seems to be that pretreatment caused a decrease in the particle size of the catalyst, with further treatment then inducing enzyme inactivation. It is noteworthy that for none of the solvents, except DMF (the most polar organic solvent), was the final activity (after ultrasonication for 4 h) less than the starting activity (without ultrasonication pretreatment).
Controls were also carried out in which the Burkholderia cepacia lipase was incubated for 4 h at 40°C (in a water bath, without irradiation) in various solvents. Here surviving activities of 75%, 100%, 100% and 53% for aqueous buffer, acetonitrile, octane and DMF respectively (data not shown) were obtained. Therefore ultrasonic pretreatment compensated effectively for thermal inactivation, with pretreatment in aqueous buffer being more effective, in terms of observed enzyme activity, than in organic solvents. Presumably, cavitation energy and its dispersion differ in aqueous and organic media.
It is relevant to compare these results with those reported by Vulfson et al  on subtilisin-catalysed transesterification. In that study a similar power output was employed (150 W), with the temperature maintained using a thermostatically-controlled glass reaction vessel. Ultrasonic treatment of subtilisin in phosphate buffer led to inactivation of the enzyme (50% in around out 2 h), while no equivalent effect was observed in t-amyl alcohol (containing 1% vv-1 buffer). Sinisterra  has also reported that the ultrasonic inactivation of subtilisin is more pronounced in aqueous media than under biphasic conditions (t-amyl alcohol, 1% phosphate buffer).
Another study of interest is that of Özbek and Ulgen , who recently investigated the effect of ultrasound on six enzymes (four dehydrogenases, alkaline phosphatase and β-galactosidase) in aqueous buffers. Apart from alkaline phosphatase, all other enzymes showed (variable) inactivation profiles (although ultrasonic treatment was carried out at 5°C). Higher ultrasonication times or power outputs resulted in greater inactivation. In addition, it was observed that on increasing the viscosity of the media by addition of glycerol increased the ultrasonic inactivation. Interestingly, none of these three studies reported enzyme activation. However, two studies by Ishimori et al. and  Sakakibara et al. , do report enhanced reaction rates resulting from the application of ultrasound to enzymatic reactions in aqueous buffers. The first of these reported the acceleration of α-chymotrypsin activity, whilst the second demonstrated that the activity of invertase was promoted by ultrasound at low substrate concentration. While the Vmax remained unaltered, Km roughly halved upon ultrasonication (i.e. affinity for the substrate increased).
Thus to recapitulate, as indicated by figure 1, ultrasonic pretreatment resulted in the aqueous enzyme being more active in aqueous buffer. These data are unusual and have not been previously observed.
The effect of ultrasonic pre-irradiation on lipase activity in organic solvents
The enhancement of initial enzymic rates is a challenge particularly relevant to industry [10, 12]. The transesterification activity of lipases has already been exploited in the synthesis and kinetic resolution of several compounds [10–12]. In order to illustrate this, we decided to look at the production of biodiesel from Jatropha oil. Biodiesel is a diesel-equivalent processed fuel consisting of short chain alkyl (methyl or ethyl) esters of fatty acids, which can be used (alone, or blended with conventional diesel fuel) in unmodified diesel-engine vehicles . It is a more environmentally-friendly fuel and its enzymatic preparation has attracted considerable attention [20–22]. The enzymatic route involves the lipase-catalysed tranesterification of plant oils with ethyl/methyl alcohol. The use of Jatropha oil as the starting material is favourable, given for instance its inedibility and easy cultivation, even on wasteland [14, 15]. Both chemical  and enzymic  preparations have been described in the literature, and earlier studies have demonstrated the benefit of a solvent-free approach [22, 23].
Structural and morphological changes in the lipase preparation as a result of ultrasonication
Limited data on the effects of ultrasonication on enzyme activity are available in the literature. While in some cases, ultrasonication results in the loss of enzymic activity, in others it leads to the enhancement of the reaction rate. In this study we demonstrated that ultrasonic pretreatment enhanced enzyme activity in water and organic media.
Jatropha oil was obtained from Dr. Jayaveera, Jawaharlal Nehru Technological University Oil Technological Research Institute, Anatapur, India. Burkholderia cepacia (PS) and Pseudomonas fluorescens (AK) were kind gifts from Amano Enzyme Inc., Nagoya, Japan. p-Nitrophenylpalmitate (pNPP) and Sephadex G-75 were bought from Sigma Chemical Co., St Louis, USA. All solvents were of anhydrous grade and were obtained from J. T. Baker, USA. They were further dried by being gentle shaken with 3 Å molecular sieves (E. Merck, Mumbai, India).
Lipase from Burkholderia cepacia (50 mg) was dissolved in 0.5 ml of 20 mM sodium phosphate buffer (pH 7.0). Pseudomonas fluorescens lipase (50 mg) was dissolved in 0.5 ml of 20 mM sodium phosphate buffer (pH 8.0). The samples were then lyophilized for 48 h. The resulting dried powders obtained were labelled as pH tuned enzymes .
Ultrasonic treatment was performed using an Elma transsonic digital ultrasonic unit (model T 490 DH) from Elma & Co. KG Hans Schmidbauer Gmbh, Singen, Germany. A fixed frequency of 40 kHz and various power ratings specified in the figure legends were employed. The ultrasonicator bath was equipped with a temperature control.
The effect of ultrasonic pre-irradiation on esterase activity
pH-tuned lipases (1 mg), each dispersed in organic solvents (100 μl) or dissolved in 20 mM of phosphate buffer (pH 7.0) (100 μl) were put into a microcentrifuge tube (1.5 ml capacity). The lipases were ultrasonicated periodically at various power ratings (110, 66, 44 W) for different time periods (i.e. 1, 2, 3 and 4 h). To avoid substantial rises in temperature, ultrasonication of cells or enzymes was not carried out continuously. Instead an 'on' and 'off' cycle was followed. In this study, we carried out ultrasonication for 5 minutes, followed by a break of 10 minutes between each cycle. This ensured that the temperature of the sample could be kept constant at 40 ± 1°C throughout. After ultrasonic irradiation, the enzyme suspensions (in organic solvents) were centrifuged at 8,000 g for 10 min at 25°C. The supernatants were removed and the lipases dissolved in 1 ml of 0.1 M sodium phosphate buffer (pH 7.0). An esterase assay using p-nitrophenyl palmitate as a substrate was performed at 37°C . One unit of enzyme activity corresponds to the production (by 1 mg of lipase) of 1 μmol of p-nitrophenol per minute . All the reactions were carried out in duplicate and the difference in the esterase activity value for each pair of duplicates was less than 6%.
Preparation of enzyme precipitated and rinsed with propanol (EPRP)  with ultrasonically pre-irradiated lipase
To dry, chilled acetone (4 ml) at 4°C was added ultrasonic pre-irradiated lipase (10 mg) in 1 ml of 20 mM phosphate buffer (pH 7.0). The precipitate was twice rinsed with 1 ml of ice-cold acetone at 4°C. Prior to use, EPRP was twice washed with 1 ml of n-octane. As a control, EPRP of untreated lipase was prepared in a similar manner .
Transesterification of ethyl butyrate with butanol
Ethyl butyrate (60 mM) and n-butanol (120 mM) were put into a vial containing 1 ml of octane, before the addition of the lipase EPRP prepared from 10 mg of lipase. The reaction mixture was incubated at 35°C at 200 rpm. Aliquots were withdrawn at different time periods and analysed by GC.
Ultrasonically pre-irradiated lipase for transesterification of Jatrophaoil
Jatropha oil (0.5 g, 600 μl) and pH tuned lipase (50 mg) were sonicated in a screw-capped vial at 110 W for different time periods (i.e. 1, 2, 3 and 4 h). In this study, we carried out ultrasonication for 5 minutes, followed by a break of 10 minutes between each cycle. This ensured that the temperature of the sample could be kept constant at 40 ± 1°C throughout. Ethanol (137 μl) was added to the ultrasonically pre-irradiated Jatropha oil/lipase mixture, and incubated at 40°C with constant shaking at 200 rpm for 24 h. The reaction was monitored by withdrawing aliquots at different time intervals and analysing them using GC. For analysis, the mixture was centrifuged and aliquots were withdrawn from the clear supernatant and diluted (5 times) with hexane. Lauric acid methyl ester was used as an internal standard. All the reactions were carried out in duplicate and the yields obtained from duplicate pairs were found to be within 5% agreement.
Gas chromatography analysis
The formation of alkyl esters was analysed on a Nucon-5700 gas chromatograph with a flame-ionisation detector. A capillary column (70% phenyl polysilphenylenesiloxane) was employed (length: 30 m; internal diameter: 0.25 mm). Nitrogen was used as the carrier gas at a constant flow rate of 4 Kg cm-2. The column oven temperature was programmed from 150 to 250°C (at the rate of 10°C min-1) with injector and detector temperatures set at 240 and 250°C, respectively.
Purification of Burkholderia cepacialipase
Burkholderia cepacia lipase was purified by gel filtration as described by Kim et al . A sample of crude lipase (2 g) was dissolved in 20 ml of 5 mM phosphate buffer (pH 7.0). To remove insoluble material, the crude enzyme solution was centrifuged at 8000 g for 20 min at 4°C. The supernatant was centrifuged again at 11,000 g for 30 min at 4°C. The clear supernatant was freeze-dried at -20°C and lyophilised for 24 h. 200 mg of the crude enzyme powder was then dissolved in 0.5 ml of 50 mM phosphate buffer and loaded on a Sephadex G-75 column (18.5 cm × 1.75 cm), which had been previously equilibrated with 5 mM of phosphate buffer (pH 7.0). Elution was performed using the same buffer. SDS/PAGE of the sample was performed as described by Hames  using a Bio-Rad Mini Orotean II electrophoresis unit and standard molecular-mass markers (Bio-Rad, Richmond, CA, USA). The gel was stained according to the silver staining method . The purified lipase was seen as a single band in SDS-PAGE.
Circular Dichroism (CD)
Far-UV CD spectra were recorded on a JASCO J-710 spectropolarimeter at 25°C over the 195–250 nm spectral range, with an optical path of 0.2 cm. The lipase concentration was 0.01 mgml-1 of 20 mM phosphate buffer (pH 7.0). Near-UV spectra were recorded over the 250–310 nm spectral range, with an optical path of 0.2 cm and lipase concentration of 1 mgml-1 of 20 mM phosphate buffer (pH 7.0).
Scanning Electron Microscopy (SEM)
SEM of samples was carried out on a Cambridge Stereoscan (S-360), Altran Corporation, Boston, USA. All the samples were dissolved in 0.1 M phosphate buffer (pH 7.0), freeze-dried and placed on the sample holder before being scanned under vacuum.
This work was supported by funds obtained from Department of Science and Technology, Govt. of India (DST).
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